Bottom Line:
Autism is a multifactorial neurodevelopmental disorder affecting more males than females; consequently, under a multifactorial genetic hypothesis, females are affected only when they cross a higher biological threshold.Here we show the use of this strategy by identifying missense and dosage sequence variants in the gene encoding the adhesive junction-associated δ-catenin protein (CTNND2) in female-enriched multiplex families and demonstrating their loss-of-function effect by functional analyses in zebrafish embryos and cultured hippocampal neurons from wild-type and Ctnnd2 mouse embryos.Finally, through gene expression and network analyses, we highlight a critical role for CTNND2 in neuronal development and an intimate connection to chromatin biology.

ABSTRACTAutism is a multifactorial neurodevelopmental disorder affecting more males than females; consequently, under a multifactorial genetic hypothesis, females are affected only when they cross a higher biological threshold. We hypothesize that deleterious variants at conserved residues are enriched in severely affected patients arising from female-enriched multiplex families with severe disease, enhancing the detection of key autism genes in modest numbers of cases. Here we show the use of this strategy by identifying missense and dosage sequence variants in the gene encoding the adhesive junction-associated δ-catenin protein (CTNND2) in female-enriched multiplex families and demonstrating their loss-of-function effect by functional analyses in zebrafish embryos and cultured hippocampal neurons from wild-type and Ctnnd2 mouse embryos. Finally, through gene expression and network analyses, we highlight a critical role for CTNND2 in neuronal development and an intimate connection to chromatin biology. Our data contribute to the understanding of the genetic architecture of autism and suggest that genetic analyses of phenotypic extremes, such as female-enriched multiplex families, are of innate value in multifactorial disorders.

Figure 4: Delta catenin is critical for maintaining functional neuronal networks(a) Gain of function: (i) Over expression of CTNND2 leads to an increase in the number of excitatory synapses. Primary dendrites from neurons transfected with GFP alone, GFP fusion with wild type CTNND2, or mutant isoforms, and immunolabeled with vGluT1 and PSD95. (ii) Quantification of number of PSD95+vGluT1 positive puncta per 100 μm of dendritic length (N=12 each). (b) Loss of function: (i) Neurons from Ctnnd2 mutants have a significant reduction in synapse density. Synapses are identified as puncta with PSD95 and vGluT1 overlap. (ii) Quantification of the number of PSD95+vGluT1 positive puncta per 100 μm of dendritic length (N=13 each). (iii) Alternatively, neurons were immunolabeled with GluA and vGluT1 to identify active functional excitatory synapses. (iv) Quantification of the number of GluA+vGluT1 positive puncta per 100 μm of dendritic length (N=15 each). (c) Rescue of loss of function: (i) WT CTNND2 but not its mutant isoforms can rescue the loss of phenotype in neurons from CTNND2 mutants. Primary dendrites from neurons transfected with GFP alone, GFP fusion with wild type CTNND2, or mutant isoforms, and immunolabeled with vGluT1 and PSD95. (ii) Quantification of the number of PSD95+vGluT1 positive puncta per 100 μm of dendritic length (N=14 each). Color used for merged panels are GFP (green) PSD95 (red), GluA (Red) and vGluT1 (blue). Student’s t-test were conducted with * and ** represents P<.05 and P<.001, respectively. Error bars represent standard error of mean.

Mentions:
Finally, we asked if these major CTNND2 sequence variants could affect neuronal circuitry by employing a well-established in vitro model system. Dendritic spines are the primary sites for excitatory synapse formation, and their dysregulation underlies many neuropsychiatric disorders16. To test if CTNND2 variants interfere with development and maintenance of spines, we prepared primary hippocampal neurons from E18 rat embryos and introduced either GFP or GFP fusion to wild type CTNND2 or to its mutant variants at DIV8. At DIV15, neurons were fixed and analyzed to assess spine density. We found that wild type CTNND2 had a significantly higher spine density versus GFP controls17. However, neurons expressing G34S had a significantly lower spine density than those expressing GFP or wild type CTNND2. Neurons expressing R713C on the other hand had the same spine density as those expressing GFP but significantly less than the one that expressed wild type CTNND2, suggesting a loss-of-function effect. In contrast, the A482T polymorphism had an effect similar to wild type CTNND2 (Extended Data Figure 8). To test if observed changes in spine density reflected changes in excitatory synapse number in the networks, we analyzed excitatory synapses i.e. overlapping region between postsynaptic marker PSD95 and presynaptic marker vGluT1 in mouse hippocampal neurons at DIV14 (Figure 4a). As with spine density, we found an increase in excitatory synapse number in neurons that overexpress wild type but not mutant CTNND2. Further, loss-of-function of CTNND2 led to a decrease in overall excitatory synapse density, as well as active synapses that express the GluA subunit of the AMPA type glutamate receptors (Figure 4b, 4c). Taken together, these results suggest that CTNND2 is critical to the formation and/or maintenance of synapses, in accord with other studies18,19. Moreover, unlike wild type CTNND2, the tested mutants failed to rescue the reduction in synapse density in CTNND2 background, demonstrating loss-of-function. Therefore, G34S and R713C impair development and/or the maintenance of mammalian neural circuitry.

Figure 4: Delta catenin is critical for maintaining functional neuronal networks(a) Gain of function: (i) Over expression of CTNND2 leads to an increase in the number of excitatory synapses. Primary dendrites from neurons transfected with GFP alone, GFP fusion with wild type CTNND2, or mutant isoforms, and immunolabeled with vGluT1 and PSD95. (ii) Quantification of number of PSD95+vGluT1 positive puncta per 100 μm of dendritic length (N=12 each). (b) Loss of function: (i) Neurons from Ctnnd2 mutants have a significant reduction in synapse density. Synapses are identified as puncta with PSD95 and vGluT1 overlap. (ii) Quantification of the number of PSD95+vGluT1 positive puncta per 100 μm of dendritic length (N=13 each). (iii) Alternatively, neurons were immunolabeled with GluA and vGluT1 to identify active functional excitatory synapses. (iv) Quantification of the number of GluA+vGluT1 positive puncta per 100 μm of dendritic length (N=15 each). (c) Rescue of loss of function: (i) WT CTNND2 but not its mutant isoforms can rescue the loss of phenotype in neurons from CTNND2 mutants. Primary dendrites from neurons transfected with GFP alone, GFP fusion with wild type CTNND2, or mutant isoforms, and immunolabeled with vGluT1 and PSD95. (ii) Quantification of the number of PSD95+vGluT1 positive puncta per 100 μm of dendritic length (N=14 each). Color used for merged panels are GFP (green) PSD95 (red), GluA (Red) and vGluT1 (blue). Student’s t-test were conducted with * and ** represents P<.05 and P<.001, respectively. Error bars represent standard error of mean.

Mentions:
Finally, we asked if these major CTNND2 sequence variants could affect neuronal circuitry by employing a well-established in vitro model system. Dendritic spines are the primary sites for excitatory synapse formation, and their dysregulation underlies many neuropsychiatric disorders16. To test if CTNND2 variants interfere with development and maintenance of spines, we prepared primary hippocampal neurons from E18 rat embryos and introduced either GFP or GFP fusion to wild type CTNND2 or to its mutant variants at DIV8. At DIV15, neurons were fixed and analyzed to assess spine density. We found that wild type CTNND2 had a significantly higher spine density versus GFP controls17. However, neurons expressing G34S had a significantly lower spine density than those expressing GFP or wild type CTNND2. Neurons expressing R713C on the other hand had the same spine density as those expressing GFP but significantly less than the one that expressed wild type CTNND2, suggesting a loss-of-function effect. In contrast, the A482T polymorphism had an effect similar to wild type CTNND2 (Extended Data Figure 8). To test if observed changes in spine density reflected changes in excitatory synapse number in the networks, we analyzed excitatory synapses i.e. overlapping region between postsynaptic marker PSD95 and presynaptic marker vGluT1 in mouse hippocampal neurons at DIV14 (Figure 4a). As with spine density, we found an increase in excitatory synapse number in neurons that overexpress wild type but not mutant CTNND2. Further, loss-of-function of CTNND2 led to a decrease in overall excitatory synapse density, as well as active synapses that express the GluA subunit of the AMPA type glutamate receptors (Figure 4b, 4c). Taken together, these results suggest that CTNND2 is critical to the formation and/or maintenance of synapses, in accord with other studies18,19. Moreover, unlike wild type CTNND2, the tested mutants failed to rescue the reduction in synapse density in CTNND2 background, demonstrating loss-of-function. Therefore, G34S and R713C impair development and/or the maintenance of mammalian neural circuitry.

Bottom Line:
Autism is a multifactorial neurodevelopmental disorder affecting more males than females; consequently, under a multifactorial genetic hypothesis, females are affected only when they cross a higher biological threshold.Here we show the use of this strategy by identifying missense and dosage sequence variants in the gene encoding the adhesive junction-associated δ-catenin protein (CTNND2) in female-enriched multiplex families and demonstrating their loss-of-function effect by functional analyses in zebrafish embryos and cultured hippocampal neurons from wild-type and Ctnnd2 mouse embryos.Finally, through gene expression and network analyses, we highlight a critical role for CTNND2 in neuronal development and an intimate connection to chromatin biology.

ABSTRACTAutism is a multifactorial neurodevelopmental disorder affecting more males than females; consequently, under a multifactorial genetic hypothesis, females are affected only when they cross a higher biological threshold. We hypothesize that deleterious variants at conserved residues are enriched in severely affected patients arising from female-enriched multiplex families with severe disease, enhancing the detection of key autism genes in modest numbers of cases. Here we show the use of this strategy by identifying missense and dosage sequence variants in the gene encoding the adhesive junction-associated δ-catenin protein (CTNND2) in female-enriched multiplex families and demonstrating their loss-of-function effect by functional analyses in zebrafish embryos and cultured hippocampal neurons from wild-type and Ctnnd2 mouse embryos. Finally, through gene expression and network analyses, we highlight a critical role for CTNND2 in neuronal development and an intimate connection to chromatin biology. Our data contribute to the understanding of the genetic architecture of autism and suggest that genetic analyses of phenotypic extremes, such as female-enriched multiplex families, are of innate value in multifactorial disorders.